Molecular modeling and description of a newly

Otto et al. Diagnostic Pathology 2012, 7:146
http://www.diagnosticpathology.org/content/7/1/146
SHORT REPORT
Open Access
Molecular modeling and description of a newly
characterized activating mutation of the EGFR
gene in non-small cell lung cancer
Claudia Otto, Agnes Csanadi, Paul Fisch, Martin Werner and Gian Kayser*
Abstract: Lung cancer is the leading cause of death among malignant diseases in humans worldwide. In the last
decade development of new targeted drugs for the treatment of non-small cell lung cancer proved to be a
promising approach to prolong the otherwise very poor prognosis of patients with advanced UICC stages.
Epidermal growth factor receptor (EGFR) has been in the focus of this lung cancer science and specific activating
mutations are eligible for the treatment with specific tyrosine kinase inhibitors like gefitinib or erlotinib. Beside
typical deletions in exon 19 and point mutations in exons 18 and 21 several insertions in exon 19 have been
described and attributed activating properties as well. This is the first European and overall the 5th description in
English literature of one of these specific insertions. To elucidate its structural changes leading to the activating
properties we performed molecular modeling studies. These revealed conformational and electrostatic force field
changes in the kinase domain of EGFR. To not miss uncommon mutations thorough and precise characterization of
EGFR hotspots, i. e. at least exons 18, 19 and 21, should therefore be conducted to provide best medical care and
to offer lung cancer patients appropriate cancer treatment.
Virtual slides: The vistual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/
2209889658102062
Short report
Mutations in the epidermal growth factor receptor (EGFR)
are of special diagnostic value in advanced non-small cell
lung cancer patients with therapeutic consequences [1-3].
The tyrosine kinase EGFR promotes via KRAS and PI3K
mediated pathways tumor cell proliferation, cell survival
and escape from apoptosis, while metabolic pathways are
shifted towards synthesis of basic cellular elements such
as nucleotids, fatty and amino acids [4-7]. Activating
mutations in the EGFR gene are located in exons 18
through 21 while more than 90% consist of deletions in
exon 19 and L858R substitution in exon 21. These activating mutations are eligible for the treatment with modern
tyrosine kinase inhibitors (TKI), e. g. gefitinib or erlotinib
[5,8,9]. Just recently, insertions in exon 19 have been
described to have an activating character and are responsive to TKI therapy. These shall comprise approximately
1% of EGFR mutations [10]. Described insertions are
located at the mutation hotspot of nucleotids 2212 to
* Correspondence: [email protected]
Institute of Pathology, University Medical Center Freiburg, Breisacher Strasse
115a, D-79106 Freiburg, Germany
2234 thus just prior to the prevalence point of exon 19
deletions [2,10-17].
In a 79 year old Kaukasian female patient we were
requested to perform EGFR mutation analysis on a liver
metastasis of a primary lung adenocarcinoma. The tissue
specimen was collected by core biopsy. Histologic examination revealed metastatic formations of a predominantly
solid adenocarcinoma. Immunohistochemical nuclear
expression of TTF1 proved this to be of primary pulmonary origin (Figure 1). After microdissection of the tumor
to enrich neoplastic DNA we performed Sanger sequencing for exons 18, 19 and 21 according to published routine protocol [18]. While exons 18 and 21 were of
wildtype sequence this analysis revealed a duplication of
18 nucleotids at position 22147_2235 (c.22147_2235dupl)
in exon 19 which leads to the insertion of 6 amino acids
(p.K745_E746insIPVAIK) (Figure 2A). Uncommon for
EGFR mutations in non-small cell lung cancer the female
patient presented here was of older age.
Since this is the 5th description of this specific duplication in exon 19 of the EGFR-gene in English literature
and to our knowledge the first in Europe molecular
© 2012 Otto et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Otto et al. Diagnostic Pathology 2012, 7:146
http://www.diagnosticpathology.org/content/7/1/146
Page 2 of 4
Figure 1 Histologic morphology of the liver metastasis of the
primary lung adenocarcinoma with predominantly solid growth
pattern. (A hematoxilin-eosin stain, 10x; B immunohistochemical
stain for TTF1, 10x).
modeling studies were performed using the SWISS-Prot
server [19,20] to investigate the structural changes of
this mutation. Crystal structures of wildtype EGFR kinase domain without ligand (protein data bank entry
2gs2) and in complex with gefitinib (protein databank
entry 1m17) served as templates for the homology modeling calculations. Visualization confirmed conformational changes in the ATP-binding pocket due to this
insertion (Figure 2B and C). Most prominent is the displacement of the side chain residue of Lysin at position
751 (K751). In the wildtype structure this polar side
chain is in the vicinity of the hydrophobic aromatic ring
of gefitinib. Our modeling results indicate that due to
the mutation the hydrophilic residue of Lysin751 opens
the binding pocket. This may result in an alteration of
electrostatic forces which could lead to a facilitated
binding of gefitinib (Figure 2B and C).
Insertions at this site of the EGFR gene result in a
change in the ATP-binding pocket allowing a facilitated
activation of effector proteins like RAS or PI3Kinase to
promote tumor cell proliferation and survival [10,21]. The
conformational changes observed here could also explain
these published capabilities of exon 19 insertions.
Since exon 19 and especially this subregion of the
exon displays a hotspot for genetic mutations of the
EGFR gene specific DNA structures at this location on
chromosome 7 must prevail. Amongst those small loops
due to an adenosine and thymidine rich DNA segment
at this location can be suspected to enable cross links in
A
B
C
K756
K751
K751
Figure 2 Molecular characterization of the described insertion in Exon 19 of the EGFR-gene. A) Sanger sequence of the mutation in exon
19 of the EGFR-gene. Indicated by the red frame are the duplicated 18 nucleotides which do not result in a mutation-typical double curve
pattern. As wildtype and mutated DNA strands differ beginning with the end of the insertion/duplication, typical double curve pattern is
occurring right behind the mutation. B) Molecular model of the mutated protein highlighting the insertion (orange area). Lysin 751 (K751, red) is
displaced away from the aromatic ring of gefitinib, thus opening the ATP-binding pocket and changing its electrostatic properties. The 6
aminoacids following the insertion (yellow area) are now in a coiled-coil tertiary structure. C) Molecular model of wildtype EGFR in complex with
gefitinib. Lysin 751 (K751, red) is in the vicinity of the aromatic ring of gefitinib. The yellow area is highlighting the site of the duplication.
Otto et al. Diagnostic Pathology 2012, 7:146
http://www.diagnosticpathology.org/content/7/1/146
between the same or the complementary DNA-strand
[22]. These cross links may then not only result in deletions, which are the most common mutations at this site
of exon 19, but also in a duplication, as described here
of 18 nucleotides.
Several methods for the evaluation of EGFR-status in
non-small cell lung cancer have been described [23-30].
Nevertheless contemporarily new mutations are being
detected and their biological impact is in the focus of
scientific research. Thus, complete sequencing of the
mutation hotspots, i. e at least EGFR exons 18, 19 and
21, leads not only to new insights in tumor biology but
also has great therapeutical impact for the patient. Only
complete sequenicing will detect new or uncommon
mutations which may also respond to TKI therapy.
Concluding, insertions in exon 19 are likely to respond
to TKI therapy. Since not only activating mutations of
the EGFR gene exist, but secondary resistance to TKItherapy can be a result of additional mutations thorough
analysis and exact characterization of EGFR mutations
by sequence analysis in non-small cell lung cancer
should be performed.
Ethics
Data publication is in concordance with the decision of
the ethics committee of the University Medical Center
Freiburg (EK 10/12).
Page 3 of 4
6.
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9.
10.
11.
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15.
16.
Competing interests
The authors state that no conflict of interest exists.
17.
Authors’ contributions
Clinical assessment and mutation analysis CO and GK; scientific workup and
molecular modeling CO, GK; manuscript preparation CO, GK; proofreading:
CO, AC, PF, GK, MW. All authors read and approved the final manuscript.
Acknowledgements
The article processing charge was funded by the German Research
Foundation (DFG) and the Albert Ludwigs University Freiburg in the funding
program Open Access Publishing.
Received: 20 September 2012 Accepted: 17 October 2012
Published: 22 October 2012
18.
19.
20.
21.
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doi:10.1186/1746-1596-7-146
Cite this article as: Otto et al.: Molecular modeling and description of a
newly characterized activating mutation of the EGFR gene in non-small
cell lung cancer. Diagnostic Pathology 2012 7:146.
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